Every day nearly 14,000 people are newly infected with HIV-1 and over 80 % of these infections are transmitted by the mucosal route during unprotected rectal or vaginal intercourse. The development of a safe and effective topical microbicide may offer a preventative strategy that could have a major impact on the course of the HIV pandemic. Six candidate topical microbicides are currently undergoing clinical effectiveness trials and many others are under development. However, potential shortcomings of topical microbicides are: the requirement of immediate pre-coital application, possible vaginal irritation/inflammation upon long-term use which may result in enhanced HIV-1 transmission, and relatively high production costs. The strategy of using live microbial microbicides may solve the potential problems inherent to chemically based microbicides. Here, we seek to develop a live microbial anti-HIV microbicide based on simian-derived vaginal Lactobacilli isolates. The cervicovaginal mucosa is primarily colonized by commensal Lactobacilli and the secretion of substantial quantities of an anti-HIV microbicide by genetically engineered Lactobacilli may effectively block vaginal transmission of HIV-1. We will identify vaginal Lactobacilli isolates from macaques which demonstrate high hydrogen peroxide (H2O2) production and strong adherence to vaginal epithelial cells, critical factors for in situ colonization. Plasmid systems will be engineered to express and secrete anti-HIV compounds. The ability of recombinant Lactobacilli strains to colonize the cervicovaginal tract will be tested in rhesus macaques. Finally, macaques will be inoculated with recombinant Lactobacillus secreting anti-HIV microbicides and subsequently challenged with chimeric/simian immunodeficiency virus SHIV-162P3 to determine protective efficacy. The goal of this application is to develop a live microbial topical microbicide against HIV-1 based on vaginal Lactobacillus isolates that may offer the following significant advantages over chemically based topical microbicides: 1) Long-lasting protection due to bacterial colonization, thus eliminating the need for direct pre-coital application and more control by the vulnerable receptive partner; 2) Low potential to induce inflammatory reactions by using vaginal commensal bacteria; and 3) Low production costs compared to protein-based microbicides. [unreadable] [unreadable] [unreadable] [unreadable]